Concise synthesis and biological activity evaluation of novel pyrazinyl-aryl urea derivatives against several cancer cell lines, which can especially induce T24 apoptotic and necroptotic cell death.

Based on the structural modification of regorafenib, 28 pyrazinyl-aryl urea derivatives were synthesized and their in vitro antiproliferative activities were evaluated. Six compounds (5-16, 5-17, 5-18, 5-19, 5-22, and 5-23) exhibited favorable inhibitory activity against the human bladder cancer T24 cell line, and 5-23 demonstrated the strongest inhibitory activity (IC50 = 4.58 ± 0.24 µM) with high selectivity. Compound 5-23 induced apoptosis in the low concentration range (≤7.5 µM) combined with shorter incubation time (≤10 h) via the activation of caspases, while high concentrations and prolonged incubation times led to necroptotic cell death by activating the RIPK1/RIPK3/MLKL signaling pathway. Induced apoptosis and necroptosis were closely associated with intracellular reactive oxygen species generation and decreased mitochondrial membrane potential. Compared with regorafenib, 5-23 displayed improved pharmacokinetic profiles in an in vivo rat model. Molecular docking and structure-activity relationship analyses were in agreement with the biological data. Compound 5-23 may be a potent anti-bladder cancer agent and this small molecule can be considered as a promising structure for further optimization.

Correction: Concise synthesis and biological activity evaluation of novel pyrazinyl-aryl urea derivatives against several cancer cell lines, which can especially induce T24 apoptotic and necroptotic cell death.

[This corrects the article DOI: 10.1039/D1MD00306B.].

Synthesis and in vitro anti-bladder cancer activity evaluation of quinazolinyl-arylurea derivatives.

Based on the structural modification of molecular-targeted agent sorafenib, a series of quinazolinyl-arylurea derivatives were synthesized and evaluated for their anti-proliferative activities against six human cancer cell lines. Compared with other cell lines tested, T24 was more sensitive to most compounds. Compound 7j exhibited the best profile with lower IC50 value and favorable selectivity. In this study, we focused on 7j-induced death forms of T24 cells and tried to elucidate the reason for its potent proliferative inhibitory activity. Compound 7j treatment could trigger three different cell death forms including apoptosis, ferroptosis, and autophagy; which form would occur depended on the concentrations and incubation time of 7j: (1) Lower concentrations within the initial 8 h of 7j treatment led to apoptosis-dependent death. (2) Ferroptosis and autophagy occurred in the case of higher concentrations combining with extended incubation time through effectively regulating the Sxc-/GPx4/ROS and PI3K/Akt/mTOR/ULK1 pathways, respectively. (3) The above death forms were closely associated with intracellular ROS generation and decreased mitochondrial membrane potential induced by 7j. In molecular docking and structure-activity relationship analyses, 7j could bind well to the active site of the corresponding receptor glutathione peroxidase 4 (GPx4). Compound 7j could be a promising lead for molecular-targeted anti-bladder cancer agents' discovery.

Hypertonic saline resuscitation protects against kidney injury induced by severe burns in rats.

BACKGROUND: Proper fluid resuscitation can relieve visceral damage and improve survival in severely burned patients. This study compared the effectiveness of resuscitation with 400mEq/L hypertonic saline (HS) and sodium lactate Ringer's solution (LR) in rats with kidney injury caused by burn trauma. METHODS: Rats (Sprague-Dawley) underwent burn injury and were randomized into sham, LR, and HS groups. Samples from the kidney were assayed for water content ratio, histopathology, and oxidative stress (superoxide dismutase (SOD) and malondialdehyde (MDA)). Serum sodium, renal function (creatinine and cystatin (Cys)-C), and inflammatory response (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and high mobility group protein box (HMGB)-1) were also examined as serum markers. RESULTS: Hypertonic saline resuscitation reduced the renal water content ratio and improved renal histopathology caused by severe burns. This effect was accompanied by reductions in serum creatinine and Cys-C as well as TNF-α, IL-1ß, and HMGB1. Serum sodium concentration and SOD activity were increased, whereas MDA content was decreased in the kidney tissue of the HS group. CONCLUSIONS: The data indicate that 400mEq/L HS solution reduces hyponatremia and renal edema, inhibits the release of inflammatory mediators, and alleviates oxidative stress injury, thus protecting against kidney injury induced by severe burns.

200mM hypertonic saline resuscitation attenuates intestinal injury and inhibits p38 signaling in rats after severe burn trauma.

BACKGROUND: An overabundant discharge of inflammatory mediators plays a significant role in intestinal injury throughout the early stages of critical burns. The present study aims to explore the outcome of 200mM hypertonic saline (HS) resuscitation on the intestinal injury of critically burned rats. MATERIALS AND METHODS: Fifty-six Sprague-Dawley rats were randomized into three groups: sham group (group A), burn plus lactated Ringer's group (group B), and burn plus 200mM HS group (group C). Samples from the intestine were isolated and assayed for wet-weight-to-dry-weight (W/D) ratio, histopathology analyses, and p38 mitogen-activated protein kinase (MAPK) activity. Serum interleukin 1ß (IL-1ß) and high mobility group protein box 1 (HMGB1) concentrations were also examined. RESULTS: Initial resuscitation with 200mM Na+ HS significantly decreased the intestinal W/D ratio and improved intestinal histopathology caused by severe burn. HS resuscitation also inhibited the increase of serum IL-1ß and HMGB1 concentrations, and p38 MAPK activity in the intestine of critically burned rats. CONCLUSIONS: The overall findings of this study suggest that preliminary resuscitation with 200mM HS after severe thermal injury reduces intestinal edema, inhibits systemic inflammatory response, and attenuates intestinal p38 MAPK activation, thus reduces burns-induced intestinal injury.

Puerarin attenuates severe burn-induced acute myocardial injury in rats.

BACKGROUND: Puerarin, the main isoflavone glycoside extracted from the root of Pueraria lobata, is widely prescribed for patients with cardiovascular disorders in China. This study investigates the effect of puerarin on severe burn-induced acute myocardial injury in rats and its underlying mechanisms. MATERIALS AND METHODS: Healthy adult Wistar rats were divided into three groups: (1) sham group, sham burn treatment; (2) burn group, third-degree burns over 30% of the total body surface area (TBSA) with lactated Ringer's solution for resuscitation; and (3) burn plus puerarin group, third-degree burns over 30% of TBSA with lactated Ringer's solution containing puerarin for resuscitation. The burned animals were sacrificed at 1, 3, 6, 12, and 24 h after burn injury. Myocardial injury was evaluated by analyzing serum creatine kinase MB fraction (CK-MB) activity and cardiac troponin T (cTNT) level. Changes in cardiomyocyte ultrastructure were also determined using a transmission electron microscope. Tumor necrosis factor (TNF)-α concentration in serum was measured by radioimmunoassay. Cardiac myeloperoxidase (MPO) activity and malondialdehyde (MDA) concentration were measured to determine neutrophil infiltration and oxidative stress in the heart, respectively. The expression of p38 mitogen-activated protein (MAP) kinase in the heart was determined by Western blot analysis. RESULTS: After the 30% TBSA full-thickness burn injury, serum CK-MB activities and cTnT levels increased markedly, both of which were significantly decreased by the puerarin treatment. The level of serum TNF-α concentration in burn group at each time-point was obviously higher than those in sham group (1.09±0.09 ng/ml), and it reached the peak value at 12 h post burn. Burn trauma also resulted in worsen ultrastructural condition, elevated MPO activity and MDA content in heart tissue, and a significant activation of cardiac p38 MAP kinase. Administration of puerarin improved the ultrastructural changes in cardiomyocytes, decreased TNF-α concentration in serum as well as suppressed cardiac MPO activity and reduced MDA content, and abolished the activation of p38 MAP kinase in heart tissue after severe burn. CONCLUSIONS: These results suggest that puerarin attenuates inflammatory responses, reduces neutrophil infiltration and oxidative stress in the heart, and protects against acute myocardial injury induced by severe burn.

[Effect of high mobility group box protein 1 on the Kupffer cells of rats with severe burn and the role of receptor for advanced glycation end products in the process].

OBJECTIVE: To investigate the effect of high mobility group box protein 1 (HMGB1) on the production of pro-inflammatory cytokines by Kupffer cell (KC) of rats with severe burn and the role of receptor for advanced glycation end products (RAGE) in the process. METHODS: Model of 30% TBSA full-thickness burn was reproduced in 32 SD rats through immersing the back in 98°C water for 12 s. KC (32 samples) was isolated from rat liver 24 h after injury and inoculated in 24-well plate in the concentration of 1×10(6) cell per well. (1) Cells were divided into control group (cultured with 1 mL PBS) and HMGB1 group (stimulated with 100 ng/mL HMGB1 in the volume of 1 mL) according to the random number table, with 8 samples in each group. At post culture hour (PCH) 48, the expression of RAGE (denoted as grey value ratio) was detected with Western blotting. (2) Another portion of cells were divided into control group (cultured with 1 mL PBS), HMGB1 group (treated with 100 ng/mL HMGB1 in the volume of 1 mL), HMGB1 + anti-RAGE antibody group (treated with 100 ng/mL HMGB1 in the volume of 1 mL after being pre-incubated with 20 µg/mL anti-RAGE monoclonal antibody in the volume of 1 mL for 2 hours), HMGB1 + recombinant rat RAGE/Fc chimera (rrRAGE/Fc) group (treated with the mixture of 100 ng/mL HMGB1 in the volume of 0.5 mL and 5 µg/mL rrRAGE/Fc in the volume of 0.5 mL which were pre-incubated for 2 hours) according to the random number table, with 8 samples in each group. At PCH 48, the protein levels of TNF-α and IL-1ß in supernatant were determined with enzyme-linked immunosorbent assay, while the mRNA expression of TNF-α and IL-1ß (denoted as grey value ratio) were determined with Northern blotting. Data were processed with one-way analysis of variance, t test, and LSD test. RESULTS: (1) The expression of RAGE in HMGB1 group (1.036 ± 0.101) was significantly higher than that of control group at PCH 48 (0.191 ± 0.024, t = -23.158, P = 0.000). (2) In HMGB1 group, HMGB1 + anti-RAGE antibody group, and HMGB1 + rrRAGE/Fc group, the contents of TNF-α in supernatant were respectively (10.59 ± 1.39), (9.91 ± 1.68), (11.51 ± 2.27) ng/mL; the contents of IL-1ß in supernatant were respectively (2.49 ± 0.33), (2.08 ± 0.32), (2.42 ± 0.42) ng/mL; the mRNA levels of TNF-α in cells were respectively 0.311 ± 0.009, 0.301 ± 0.047, 0.326 ± 0.016; the mRNA levels of IL-1ß in cells were respectively 0.237 ± 0.021, 0.244 ± 0.041, 0.245 ± 0.013. There were no statistically significant differences in the above indexes among these three groups (with P values all above 0.05). Their levels were all significantly higher than those of control group [with contents of TNF-α and IL-1ß in supernatant respectively (2.69 ± 0.14), (0.43 ± 0.05) ng/mL, and mRNA levels of TNF-α and IL-1ß in cells respectively 0.140 ± 0.022, 0.077 ± 0.005, P values all below 0.01]. CONCLUSIONS: HMGB1 can induce the production of pro-inflammatory cytokines TNF-α and IL-1ß from the KC in rats with severe burn. However, RAGE does not play a predominant role in this process.

Sodium butyrate protects against severe burn-induced remote acute lung injury in rats.

High-mobility group box 1 protein (HMGB1), a ubiquitous nuclear protein, drives proinflammatory responses when released extracellularly. It plays a key role as a distal mediator in the development of acute lung injury (ALI). Sodium butyrate, an inhibitor of histone deacetylase, has been demonstrated to inhibit HMGB1 expression. This study investigates the effect of sodium butyrate on burn-induced lung injury. Sprague-Dawley rats were divided into three groups: 1) sham group, sham burn treatment; 2) burn group, third-degree burns over 30% total body surface area (TBSA) with lactated Ringer's solution for resuscitation; 3) burn plus sodium butyrate group, third-degree burns over 30% TBSA with lactated Ringer's solution containing sodium butyrate for resuscitation. The burned animals were sacrificed at 12, 24, and 48 h after burn injury. Lung injury was assessed in terms of histologic changes and wet weight to dry weight (W/D) ratio. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 protein concentrations in bronchoalveolar lavage fluid (BALF) and serum were measured by enzyme-linked immunosorbent assay, and HMGB1 expression in the lung was determined by Western blot analysis. Pulmonary myeloperoxidase (MPO) activity and malondialdehyde (MDA) concentration were measured to reflect neutrophil infiltration and oxidative stress in the lung, respectively. As a result, sodium butyrate significantly inhibited the HMGB1 expressions in the lungs, reduced the lung W/D ratio, and improved the pulmonary histologic changes induced by burn trauma. Furthermore, sodium butyrate administration decreased the TNF-α and IL-8 concentrations in BALF and serum, suppressed MPO activity, and reduced the MDA content in the lungs after severe burn. These results suggest that sodium butyrate attenuates inflammatory responses, neutrophil infiltration, and oxidative stress in the lungs, and protects against remote ALI induced by severe burn, which is associated with inhibiting HMGB1 expression.

Effect of 200 mEq/L Na+ hypertonic saline resuscitation on systemic inflammatory response and oxidative stress in severely burned rats.

BACKGROUND: Excessive release of inflammatory mediators and oxidative stress play important roles in the increased vascular permeability and systemic edema during the early stage of severe burn. This study investigates the effect of 200 mEq/L Na(+) hypertonic saline (HS) on systemic inflammatory response and oxidative stress in severely burned rats. MATERIALS AND METHODS: Sprague-Dawley rats were divided into three groups: sham group, burn plus lactated Ringer's group, and burn plus HS group. Lung edema was assessed in terms of wet-weight-to-dry-weight ratio. Tumor necrosis factor α and interleukin 6 concentrations in serum were examined by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells were isolated and the expression of p38 mitogen-activated protein kinase was determined by Western blot analysis. The lung and intestinal concentrations of malondialdehyde, an indicator of oxidative stress, were also measured. RESULTS: Resuscitation with 200 mEq/L Na(+) HS significantly decreased the lung wet-weight-to-dry-weight ratio and abolished hyponatremia induced by burn injury. HS treatment also prevented the increases of myeloperoxidase activity and malondialdehyde content in the lung and intestine of severely burned rats. However, there were no significant differences, either in serum tumor necrosis factor α and interleukin 6 concentrations or with respect to the p38 mitogen-activated protein kinase expression in peripheral blood mononuclear cells, between the burn plus lactated Ringer's group and burn plus HS group (P > 0.05). CONCLUSIONS: Initial resuscitation with 200 mEq/L Na(+) HS after severe burn injury decreases pulmonary edema, prevents hyponatremia, and attenuates oxidative stress, but is not capable of inhibiting the systemic inflammatory response.

High-mobility group box-1 induces proinflammatory cytokines production of Kupffer cells through TLRs-dependent signaling pathway after burn injury.

Kupffer cells (KCs) were a significant source of cytokine release during the early stage of severe burns. High mobility group box protein 1 (HMGB1) was recently identified as a new type of proinflammatory cytokine. The ability of HMGB1 to generate inflammatory responses after burn trauma has not been well characterized. KCs were isolated from sham animals and rats with a 30% full-thickness burn, and then were stimulated with increasing concentrations of HMGB1. The levels of Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in culture supernatant were measured by enzyme-linked immunosorbent assay. Northern blot analysis was performed to detect the expressions of TNF-α and IL-1ß mRNAs. The activities of p38 MAPK and JNK (by Western blot analysis) as well as NF-κB (by EMSA) in KCs were also examined. As a result, HMGB1 in vitro upregulated expressions of TNF-α and IL-1ß of KCs in a dose-dependent manner, and HMGB1 promoted KCs from burn rats to produce significantly more TNF-α and IL-1ß proteins than those from sham animals. After harvested from burn rats, KCs were pre-incubated with anti-TLR2 or anti-TLR4 antibody prior to HMGB1 administration. HMGB1 exposure not only significantly increased expressions of TNF-α and IL-1ß mRNAs in KCs from burn rats, but also enhanced activities of p38 MAPK, JNK and NF-κB. However, these upregulation events were all reduced by pre-incubation with anti-TLR2 or anti-TLR4 antibody. These results indicate that HMGB1 induces proinflammatory cytokines production of KCs after sever burn injury, and this process might be largely dependent on TLRs-dependent MAPKs/NF-κB signal pathway.

Role of angiotensin II type 1 receptor in angiotensin II-induced cytokine production in macrophages.

This study was aimed to investigate the regulatory effects of angiotensin II (Ang II) on the expression of Ang II type 1 (AT1) receptor and to characterize the mechanism underlying AT1 receptor promotion of inflammatory cytokines and reactive oxygen species (ROS). The RAW 264.7 cells were stimulated with various concentrations of Ang II for different times. The mRNA and protein expressions of AT1 receptor in the cells were determined by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. The macrophages secretion of proinflammatory cytokines tumor necrosis factor-α, interleukin-1ß (IL-1ß), IL-6, and anti-inflammatory cytokine IL-10 and generation of ROS (by flow cytometry) were also examined. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activities were determined by electrophoretic mobility shift assay. Ang II resulted in an upregulation of AT1 receptor expression in dose- and time-dependent manners in macrophages. Ang II not only induced the production of tumor necrosis factor-α, IL-1ß, IL-6, and IL-10 but also increased the release of ROS. Further, both NF-κB and AP-1 were activated after stimulation with Ang II. However, these events were all abolished by preincubation with ZD7155, a selective competitive antagonist for the AT1 receptor. These results suggest that the AT1 receptor plays an important role in Ang II-induced cytokines production and ROS release via NF-κB and AP-1 pathways in macrophages.